This invention relates to a novel crystalline diphosphate salt of (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[[2,6-dideoxy-3-C-methyl-3-0-methyl-4-C-[(propylamino)methyl]-xcex1-L-ribo-hexopryanosyl]oxy]-2-ethyl-3,4,10-trihydroxy-3,5,8,10,12,14-hexamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-xcex2-D-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one (hereinafter the diphosphate salt) that is useful as an antibacterial and antiprotozoal agent in mammals. This invention also relates to pharmaceutical compositions containing the free base of the diphosphate salt and the methods of treating bacterial and protozoal infections in mammals by administering the free base of the diphosphate salt to mammals requiring such treatment. The free base of the diphosphate salt of the present invention possesses potent activity against various bacterial and protozoal infections when given by parenteral application to mammals.
Macrolide antibiotics are known to be useful in the treatment of a broad spectrum of bacterial and protozoal infections in mammals, fish and birds. Such antibiotics include various derivatives of erythromycin A such as azithromycin which is commercially available and is referred to in U.S. Pat. Nos. 4,474,768 and 4,517,359 both of which are incorporated herein by reference in their entirety.
The present invention relates to two polymorphs of (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[[2,6-dideoxy-3-C-methyl-3-0-methyl-4-C[(propylamino)methyl]-xcex1-L-ribo-hexopryanosyl]oxy]-2-ethyl-3,4,10-trihydroxy-3,5,8,10,12,14-hexamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-xcex2-D-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one diphosphate (hereinafter the diphosphate salt) shown below: 
Wherein n is 0 to 8.
In one embodiment of the invention, the diphosphate salt is a liquid crystal that has a lath-like habit and exhibits longitudinal cleavage but no lateral cleavage. X-ray diffraction shows little or no order.
In another embodiment, the disphosphate salt is crystalline. Microscopy of the diphosphate salt indicates a plate or prism habit, both of which are highly birefringent. The diphosphate salt is a well-ordered crystal characterized by the X-ray diffraction pattern below:
The crystalline diphosphate salt gradually absorbs water to about 13% at 87% relative humidity with a rapid uptake of water to about 48% occurring at about 90% relative humidity. Thermogravametric analysis on the diphosphate salt discloses that three waters are lost by about 75xc2x0 C., a fourth water is lost by about 120xc2x0 C., the fifth water is lost by about 170xc2x0 C., and the final three waters are lost by about 200xc2x0 C. The maximum number of water molecules necessary for the stabilization of the crystalline lattice appears to be about eight. Drying the diphosphate salt in about 70xc2x0 C. in air or about 45xc2x0 C. in a vacuum removed the water leaving a pseudomorph. The crystalline diphosphate salt has an aqueous solubility of about 280mg/mL.
A method of preparing the diphosphate salt comprises dissolving the anhydrous free base with magnetic stirring in absolute ethanol at ambient temperature, adding a solution of phosphoric acid in absolute ethanol over about 2 to 5 minutes to afford a precipitate, then partially dissolving the resulting solid in a small proportion of water. Stirring for several hours at ambient temperature affords a crystal crop that is collected by filtration then rinsed with several small portions of about 10/1 (v/v) ethanol-water. After the diphosphate salt has been synthesized it is reconverted to a pharmaceutically acceptable free base by a method which comprises dissolution in water, addition of methylene chloride, increasing the pH to about 8.5 to 10, collection and concentration of the organic phase, and crystallization of the amorphous free base from a hydrocarbon solvent.
A pharmaceutical composition which has antibacterial and antiprotozoal activity in mammals comprises the free base of the diphosphate salt in an amount effective in the treatment of bacterial and protozoal diseases and a pharmaceutically acceptable carrier. A method of treating bacterial and protozoal infections comprises administering to mammals in need of such treatment an antibacterial amount of the free base of the diphosphate salt. The antibacterial amount of the free base is given by parenteral application to mammals.
The present invention relates to two polymorphs of (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[[2,6-dideoxy-3-C-methyl-3-0-methyl-4-C-[(propylamino)methyl]-xcex1-L-ribo-hexopryanosyl]oxy]-2-ethyl-3,4,10-trihydroxy-3,5,8,10,12,14-hexamethyl-11-[[3,4,6-trideoxy-3-(dimethylamino)-xcex2-D-xylo-hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan-15-one diphosphate shown below: 
wherein n is 0 to 8.
In one embodiment of the invention, the diphosphate salt is a liquid crystal that has a lath-like habit and exhibits longitudinal cleavage but no lateral cleavage. X-ray diffraction shows little or no order.
In another embodiment, the diphosphate salt is a crystalline hygroscopic salt. Microscopy of the diphosphate salt indicates a plate or prism habit both of which are highly birefringent. The diphosphate salt is a well ordered crystal characterized by the X-ray diffraction pattern below:
The invention also relates to processes for preparing the liquid crystal and the crystalline diphosphate salt as well as the pharmaceutical composition of the free base of the diphosphate salt as shown in Scheme 1 below: 
Wherein n is 0-8.
Differential scanning calorimetry of the crystalline diphosphate salt shows a single event at about 119xc2x0 C. which coincides with water loss as well as the dissolution of the salt in the released water. No crystallization follows the event.
Drying in the VTI at 20-25xc2x0 C. indicated a weight loss of 10%; the dried salt gradually adsorbed water to 15% at 85% RH (relative humidity), followed by a rapid uptake to 48% at 90% RH. At 90% RH, the compound deliquesced.
The diphosphate salt was also studied by thermogravametic analysis (TGA). On samples rehydrated at 87% RH [13% water by Karl Fisher (KF) titration], four distinct breaks were noted. There were 3 waters lost by about 75xc2x0 C.; 4 by about 120xc2x0 C., 5 by about 170xc2x0 C., and 8 lost by about 200xc2x0 C. The maximum water molecules necessary for the stabilization of the crystalline lattice appears to be eight. A reduction in the water level of the hydrate caused no change in the X-ray diffraction data indicating the formation of a pseudomorph of the hydrate.
The mobility of the water within the crystalline lattice is directly related to the relative humidity of the environment the diphosphate salt comes in contact with. For example, at about 87% relative humidity, the compound contains about 13% water; at 60% relative humidity the compound contains about 8% water; and at 40% relative humidity the diphosphate salt contains about 5% water (all calculations by KF).
Drying the diphosphate salt at 70xc2x0 C. in air or at 45xc2x0 C. under vacuum removed all the water leaving a psuedomorph. Rehydrating the salt at about 87% relative yielded the octahydrate form.